This invention relates to novel cyclic compounds which have the ability to modulate the activity of G protein-coupled receptors. The invention provides both agonists and antagonists. In preferred embodiments, the invention provides cyclic peptidic and cyclic or non-cyclic non-peptidic antagonists or agonists of C5a. The compounds of the invention are both potent and selective, and are useful in the treatment of a variety of inflammatory conditions.
Activation of human complement, a system of plasma proteins involved in immunological defence against infection and injury, contributes significantly to the pathogenesis of numerous acute and chronic diseases. In particular, the complement protein C5a has been extensively investigated. For general reviews, see Whaley (1987), and Sim (1993). Table 1 provides a summary of known roles of C5a in disease.
During host defence, the complement system of plasma proteins initiates inflammatory and cellular immune responses to stimuli such as infectious organisms (bacteria, viruses, parasites), chemical or physical injury, radiation or neoplasia. Complement is activated through a complex cascade of interrelated proteolytic events that produce multiple bioactive peptides, some of which (eg. anaphylatoxins C3a and C5a) interact with cellular components to propagate inflammatory processes. Complement activation, either by the classical pathway, after antigen-antibody (Ag/Ab) binding, or by the antibody-independent alternate pathway, ends with a terminal sequence in which protein C5 is proteolytically cleaved by C5 convertase to C5a and C5b. The latter facilitates assembly of a xe2x80x9cmembrane attack complexxe2x80x9d that punches holes in membranes of target cells such as bacteria, leading to leakage, lysis and cell death. Steps in the cascade are tightly regulated to avoid stepwise amplification of proteolysis by sequentially formed proteases. If these regulatory mechanisms become inefficient, protracted activation of complement can result, causing enhanced inflammatory responses as in autoimmune diseases.
Although the broad features of the complement system and its activation are known, mechanistic details remain poorly understood. A principal and very potent mediator of inflammatory responses is the plasma glycoprotein C5a, which interacts with specific surface receptors (C5aR) on mast cells, neutrophils, monocytes, macrophages, non-myeloid cells, and vascular endothelial cells (Gerard and Gerard, 1994). C5aR is a G protein-coupled receptor with seven transmembrane helices (Gerard and Gerard, 1991). This receptor is one of the rhodopsin superfamily of GTP-linked binding proteins, but differs from rhodopsin receptors in that the receptor and G protein are linked prior to activation.
G protein-coupled receptors are prevalent throughout the human body, comprising approximately 80% of known cellular receptor types, and mediate signal transduction across the cell membrane for a very wide range of endogenous ligands. They participate in a diverse array of physiological and pathophysiological processes, including, but not limited to those associated with cardiovascular, central and peripheral nervous system, reproductive, metabolic, digestive, immunoinflammatory, and growth disorders, as well as other cell-regulatory and proliferative disorders. Agents, both agonists and antagonists, which selectively modulate functions of G protein-coupled receptors have important therapeutic applications.
C5a is one of the most potent chemotactic agents known, and recruits neutrophils and macrophages to sites of injury, alters their morphology; induces degranulation; increases calcium mobilisation, vascular permeability (oedema) and neutrophil adhesiveness; contracts smooth muscle; stimulates release of inflammatory mediators (including histamine, TNF-xcex1, IL-1, IL-6, IL-8, prostaglandins, leukotrienes) and lysosomal enzymes; promotes formation of oxygen radicals; and enhances antibody production (Gerard and Gerard, 1994). Overexpresion or underregulation of C5a is implicated in the pathogenesis of immunoinflammatory conditions such as rheumatoid arthritis, adult respiratory distress syndrome (ARDS), systemic lupus erythematosus, tissue graft rejection, ischaemic heart disease, reperfusion injury, septic shock, psoriasis, gingivitis, atherosclerosis, Alzheimer""s disease, lung injury and extracorporeal post-dialysis syndrome, and in a variety of other conditions, as summarised in Table 1.
New agents which limit the pro-inflammatory actions of C5a have potential for inhibiting chronic inflammation, and its accompanying pain and tissue damage. For these reasons, molecules which prevent C5a binding to its receptors are useful for treating chronic inflammatory disorders driven by complement activation. Importantly, such compounds provide valuable new insights to mechanisms of complement-mediated immunity.
In another context, agonists of C5a receptors or other G protein-coupled receptors may also be found to have therapeutic properties in conditions either where the G protein-coupled receptor can be used as a recognition site for drug delivery, or where triggering of such receptors can be used to stimulate some aspect of the human immune system, for example in the treatment of cancers, viral or parasitic infections.
One approach to the development of agonists or antagonists of C5a is through receptor-based design, using knowledge of the three-dimensional structures of C5a, its receptor C5aR, and the interactions between them. The structure of the receptor is unknown. The solution structure of human C5a, a 74 amino acid peptide that is highly cationic and N-glycosylated with a 3 KDa carbohydrate at Asn64, has been determined and is essentially a 4-helix bundle. The C-terminal end (residues 65-74, C5a65-74) was found to be unstructured (Zuiderweg et al, 1989) and this conformational flexibility in the C-terminus has made structure-function studies extremely difficult to interpret.
C5a has a highly ordered N-terminal core domain (residues 1-64; C5a1-64), consisting of a compact antiparallel 4-helix bundle (residues 4-12, 18-26, 32-39, 46-63) connected by loops (13-17, 27-31, 40-45), and further stabilised by 3 disulphide bonds (C21-Cys47, Cys22-Cys54, Cys34-Cys55).
Although the structure of the C5a receptor, C5aR, is unknown, the C5a-binding subunit of human monocyte-derived C5aR has been cloned and identified as a G protein-coupled receptor with transmembrane helices (Gerard and Gerard, 1991). Interactions between C5a and C5aR have been the subject of many investigations which, in summary, suggest that C5a binds via a two-site mechanism in which the N-terminal core domain of C5a is involved in receptor-recognition and binding, while the C-terminus is responsible for receptor activation. This mechanism is illustrated schematically in FIG. 1. The C-terminal xe2x80x9ceffectorxe2x80x9d region alone possesses all the information necessary for signal transduction, and is thought to bind in the receptor""s interhelical region (Siciliano et al, 1994; deMartino et al, 1995).
An N-terminal interhelical positively-charged region of C5a is responsible for receptor recognition and binding, and binds to a negatively-charged extracellular domain of C5aR (site 1), while the C-terminal xe2x80x9ceffectorxe2x80x9d region of C5a is thought to bind with the interhelical region of the receptor (site 2), and is responsible for receptor activation leading to signal transduction (Siciliano et al, 1994).
Numerous short peptide derivatives of the C-terminus of C5a have been found to be agonists of C5a (Kawai et al, 1991; Kawai et al, 1992; Kohl et al, 1993; Drapeau et al, 1993; Ember et al, 1992; Sanderson et al, 1994; Sanderson et al, 1995; Finch et al, 1997; Tempero et al, 1997; Konteatis et al, 1994; DeMartino et al, 1995). The structures of some of these agonists are shown in Table 2 below (compounds 1-6). High molecular weight polypeptide inhibitors of the action of C5a at its receptor, such as monoclonal antibodies to the C5a receptor, are also known (Morgan et al, 1992).
A small molecule, N-methylphenylalanine-lysine-proline-D-cyclohexylalanine-tryptophan-D-arginine (7, MeF-Kxe2x80x94P-dCha-Wxe2x80x94R), is a full antagonist of the C5a receptor, with no agonist activity when tested on isolated cellular membranes (Konteatis et al, 1994) or intact whole cells. This hexapeptide was developed by modifications of the agonist NMe-Fxe2x80x94Kxe2x80x94P-dCha-L-r, in which the molecule was progressively substituted at leucine residues with substituents of increasing size (Cha, F, Nph and W). This had the effect of reducing agonist activity. Receptor-binding assays, performed on isolated human neutrophil membranes, showed that the antagonist had only 0.04% relative affinity of C5a for the receptor (Konteatis et al, 1994). A key feature of these reports is the definition of the binding of 7 to the C5a receptor. These authors state that the C-terminal arginine is essential for receptor binding and antagonist activity. This is also the case in all the reports of agonist activity by small peptide analogues of the C-terminus of C5a. However, for the antagonist 7, the authors go further and state that
xe2x80x9cthe C-terminal carboxylate is an essential requirement for antagonist activity and receptor binding.xe2x80x9d
They proposed that the requirement of the carboxylate is probably the result of its specific interaction with an arginine (Arg 206) in the receptor (De Martino et al, 1995). This idea was supported by a great reduction in receptor-affinity for an analogue of 7 in which the D-arginine (NH2xe2x80x94CH(CO2H)xe2x80x94(CH2)3NHC(:NH)NH2) was replaced by agmatine (NH2xe2x80x94CH2xe2x80x94(CH2)3NHC(:NH)NH2). In summary, De Martino et al claim that the D-arginine interacts via its guanidinium side chain with a negatively-charged amino acid side chain in the receptor. A second interaction between the negatively-charged C-terminal carboxylate of 7 and a positively-charged side chain residue in the receptor is also thought to occur.
We have now determined the solution structure of this hexapeptide 7 and several analogues, and have surprisingly found that in fact a terminal carboxylate group is not required for binding to C5aR or for antagonist activity, and that instead an unusual hitherto unrecognised structural feature, a turn conformation, is responsible for C5a antagonist or agonist binding and activity. The hexapeptide and several new structurally related antagonists have been examined for both their receptor-binding affinities and antagonist activity, using intact polymorphonuclear (PMN) cells. Our results show the hitherto unknown specific structural requirement for the binding of C5a antagonists or agonists to the C5a receptor, which we believe to be common to ligands for the G protein-coupled receptor family. Our establishment of this specific structural requirement has enabled us to design and develop improved molecular probes of the complement system and of C5a-based drugs, and to design small molecules that target other G protein-coupled receptors, which are becoming increasingly recognised as important drug targets due to their crucial roles in signal transduction (G protein-coupled Receptors, IBC Biomedical Library Series, 1996).
Thus our results have enabled us to design constrained structural templates which enable hydrophobic groups to be assembled into a hydrophobic array for interaction with a G protein-coupled receptor, for example at Site 2 of the C5a receptor illustrated in FIG. 1. Such templates or scaffolds, which may be cyclic or acyclic, have not heretofore been suggested for modulators of the activity of C5a receptors or other G protein-coupled receptors.
The invention provides cyclic and non-cyclic modulators of the activity of G-protein-coupled receptors.
According to a first aspect, the invention provides a compound which is an antagonist, of a G protein-coupled receptor, which has no agonist activity, and which has a cyclic or constrained acyclic structure adapted to provide a framework of approximate dimensions as follows: 
where the numerals refer to distances between Cn carbons of amino acids or their analogues or derivatives, and A, B, C and D are not necessarily on adjacent amino acids, or analogues or derivatives thereof; and
where the critical amino acid side chains are designated by A, B, C and D, or are as defined below;
A is any common or uncommon, basic, charged amino acid side chain which serves to position a positively charged group in this position, including, but not limited to the following side chains and other mimetics of arginine side chains: 
where
X is NCN, NNO2, CHNO2 or NSO2NH2;
n is an integer from 1 to 4, and
R is H or an alkyl, aryl, CN, NH2 or OH group
B is any common or uncommon aromatic amino acid side chain which serves to position an aromatic side-chain group in this position, including but not limited to the indole, indole methyl, benzyl, phenyl, naphthyl, naphthyl methyl, cinnamyl group, or any other derivatives of these aromatic groups;
C is any common or uncommon hydrophobic amino acid side chain which serves to position any alkyl, aromatic or other group in this position, including, but not limited to D- or L-cyclohexyl alanine (Cha), leucine, valine, isoleucine, phenylalanine, tryptophan, or methionin
D is any common or uncommon aromatic amino acid which serves to position an aromatic side-chain in this position, and has the structure: 
where Z includes but is not limited to indole, indole methyl, benzyl, benzene, naphthyl, naphthyl methyl, or any other derivatives of these aromatic groups, and
R1 is H or any alkyl, aromatic, acyl or aromatic-acyl group including, but not limited to methyl, ethyl, propyl, butyl, xe2x80x94COxe2x80x94CH2CH3, xe2x80x94COxe2x80x94CH3, xe2x80x94COxe2x80x94CH2CH2CH3, xe2x80x94COxe2x80x94CH2Ph, or xe2x80x94CO-Ph.
Preferably the G protein-coupled receptor is a C5a receptor.
Other cyclic or constrained acyclic molecules, which may be peptidic or non-peptide in nature, can similarly be envisaged to support groups such as A, B, C and D for interaction with a C5a receptor or other G protein-coupled receptor.
In one preferred embodiment, the compound has antagonist activity against C5aR, has no C5a agonist activity, and has the general formula: 
where A is H, alkyl, aryl, NH2, NHalkyl, N(alkyl)2, NHaryl or NHacyl; OH, Oalkyl, Oaryl.
B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or the side chain of a D- or L-amino acid selected from phenylalanine, homophenylalanine, tryptophan, homotryptophan, tyrosine, and homotyrosine;
C is the side chain of a D-, L- or homo-amino acid selected from the group consisting of proline, alanine, leucine, valine, isoleucine, arginine, histidine, aspartate, glutamate, glutamine, asparagine, lysine, tyrosine, phenylalanine, cyclohexylalanine, norleucine, tryptophan, cysteine and methionine;
D is the side chain of a D- or L-amino acid selected from the group consisting of cyclohexylalanine, homocyclohexylalanine, leucine, norleucine, homoleucine, homonorleucine and tryptophan;
E is the side chain of a D- or L-amino acid selected from the group consisting of tryptophan and homotryptophan;
F is the side chain of a D- or L-amino acid selected from the group consisting of arginine, homoarginine, lysine and homolysine; and
X1 is xe2x80x94(CH2)nNHxe2x80x94 or (CH2)nxe2x80x94Sxe2x80x94, where n is an integer of from 1 to 4, preferably 2 or 3, xe2x80x94(CH2)2Oxe2x80x94, xe2x80x94(CH2)3Oxe2x80x94, xe2x80x94(CH2)3xe2x80x94, xe2x80x94(CH2)4xe2x80x94, or xe2x80x94CH2COCHRNHxe2x80x94, where R is the side chain of any common or uncommon amino acid.
For the purposes of this specification, the term xe2x80x9calkylxe2x80x9d is to be taken to mean a straight, branched, or cyclic, substituted or unsubstituted alkyl chain of 1 to 6, preferably 1 to 4 carbons. Most preferably the alkyl group is a methyl group. The term xe2x80x9cacylxe2x80x9d is to be taken to mean a substituted or unsubstituted acyl of 1 to 6, preferably 1 to 4 carbon atoms. Most preferably the acyl group is acetyl. The term xe2x80x9carylxe2x80x9d is to be understood to mean a substituted or unsubstituted homocyclic or heterocyclic aryl group, in which the ring preferably has 5 or 6 members.
A xe2x80x9ccommonxe2x80x9d amino acid is a L-amino acid selected from the group consisting of glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine and histidine.
An xe2x80x9cuncommonxe2x80x9d amino acid includes, but is not restricted to, D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), ortho-, meta- or para-aminobenzoic acid, ornithine, citrulline, norleucine, xcex3-glutamic acid, aminobutyric acid and xcex1,xcex1-disubstituted amino acids.
For the purposes of this specification it will be clearly understood that the word xe2x80x9ccomprisingxe2x80x9d means xe2x80x9cincluding but not limited toxe2x80x9d, and that the word xe2x80x9ccomprisesxe2x80x9d has a corresponding meaning.
According to a second aspect, of the invention provides a compound which is an agonist of G protein-coupled receptors, and which has structure III 
where the numerals refer to distances between Cn carbons of amino acids or their analogues or derivatives, and A, B, C and D are not necessarily on adjacent amino acids, or analogues or derivatives thereof; and
where B is a non-aromatic amino acid, and is preferably the D- or L-form of alanine, leucine, valine, norleucine, glutamic acid, aspartic acid, methionine, cysteine, isoleucine, serine or threonine,
and A, C and D are as defined above.
Preferably the compound is of structure IV, 
where E is any amino acid other than tryptophan and homotryptophan, for example D- or L-forms of alanine, leucine, valine, norleucine, phenylalanine, glutamic acid, aspartic acid, methionine, cysteine, isoleucine, serine, threonine, and F and X1 are as defined in Structure II. Preferably the compound is an agonist of C5a.
According to a third aspect, the invention provides a composition, comprising a compound according to the invention together with a pharmaceutically-acceptable carrier or excipient.
The compositions of the invention may be formulated for oral or parenteral use, but oral formulations are preferred. It is expected that most if not all compounds of the invention will be stable in the presence of digestive enzymes. Such stability can readily be tested by routine methods known to those skilled in the art.
Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known textbooks such as Remington; The Science and Practice of Pharmacy, Vol. II, 1995 (19th edition), A. R. Gennaro (ed), Mack Publishing Company, Easton, Pa., or Australian Prescription Products Guide, Vol. 1, 1995 (24th edition) J. Thomas (ed), Australian Pharmaceutical Publishing Company Ltd, Victoria, Australia.
In a fourth aspect, the invention provides a method of treatment of a pathological condition mediated by a G protein-coupled receptor, comprising the step of administering an effective amount of a compound of the invention to a mammal in need of such treatment.
Preferably the condition mediated by a G protein-coupled receptor is a condition mediated by a C5a receptor, and more preferably involves overexpression or underregulation of C5a. Such conditions include but are not limited to rheumatoid arthritis, adult respiratory distress syndrome (ARDS), systemic lupus erythematosus, tissue graft rejection, ischaemic heart disease, reperfusion injury, septic shock, psoriasis, gingivitis, atherosclerosis, Alzheimer""s disease, lung injury and extracorporeal post-dialysis syndrome.
While the invention is not in any way restricted to the treatment of any particular animal or species, it is particularly contemplated that the compounds of the invention will be useful in medical treatment of humans, and will also be useful in veterinary treatment, particularly of companion animals such as cats and dogs, livestock such as cattle, horses and sheep, and zoo animals, including large bovids, felids, ungulates and canids.
The compounds may be administered at any suitable dose and by any suitable route. Oral administration is preferred because of its greater convenience and acceptability. The effective dose will depend on the nature of the condition to be treated, and the age, weight, and underlying state of health of the individual treatment. This will be at the discretion of the attending physician or veterinarian. Suitable dosage levels may readily be determined by trial and error experimentation, using methods which are well-known in the art.